Self-assembled DNA nanostructure containing oncogenic miRNA-mediated cell proliferation by downregulation of FOXO1 expression

FOXO1 transcription factor not only limits the cell cycle progression but also promotes cell death as a tumor suppressor protein. Though the expression of FOXO1 is largely examined in breast cancer, the regulation of FOXO1 by miRNA is yet to be explored. In the current study, self-assembled branched DNA (bDNA) nanostructures containing oncogenic miRNAs were designed and transfected to the MCF7 cell line to decipher the FOXO1 expression. bDNA containing oncogenic miRNAs 27a, 96, and 182 synergistically downregulate the expression of FOXO1 in MCF7 cells. The down-regulation is evident both in mRNA and protein levels suggesting that bDNA having miRNA sequences can selectively bind to mRNA and inhibit translation. Secondly, the downstream gene expression of p21 and p27 was also significantly downregulated in presence of miR-bDNA nanostructures. The cell proliferation activity was progressively increased in presence of miR-bDNA nanostructures which confirms the reduced tumor suppression activity of FOXO1 and the downstream gene expression. This finding can be explored to design novel bDNA structures which can downregulate the tumor suppressor proteins in normal cells and induce cell proliferation activity to identify early-phase markers of cancer

miRNA-mediated alteration of sulfatase modifying factor 1 expression using self-assembled branched DNA nanostructures

Sulfatase enzymes catalyze sulfate ester hydrolysis, thus deficiencies of sulfatases lead to the accumulation of biomolecules resulting in several disorders. One of the important sulfatases is estrone sulfatase that converts inactive estrone sulfate to active estradiol. Posttranslational modification of highly conserved cysteine residue leads to unique formylglycine in the active site of sulfatases being critical for its catalytic activity. The essential factor responsible for this modification of sulfatase is Sulfatase-Modifying Factor 1 (SUMF1). The role of estrone sulfatase is well evident in breast cancer progression. However, the function and regulation of SUMF1 in cancer are not studied. In the present study, for the first time, we have assessed the expression of SUMF1 in breast cancer and report the oncogenic behavior upon overexpression of SUMF1. Although increased expression or activity of SUMF1 is anticipated based on its function, the expression of SUMF1 was found to be reduced in breast cancer cells at both mRNA and protein levels. An estrogen receptor (ER) dependent expression of SUMF1 was observed and higher SUMF1 expression is associated with improved breast cancer patient survival in ER-positive cases. However, high SUMF1 expression leads to reduced median survival in ER-negative breast cancer patients. Putative binding sites for miRNAs-106b-5p, 128-3p and 148b-3p were found at 3′-UTR of SUMF1. Since self-assembled branched DNA (bDNA) structures have emerged as a highly efficient strategy for targeting multiple miRNAs simultaneously, we studied the alteration in SUMF1 expression using bDNA nanostructures with a complementary sequence to miRNAs. The findings suggest the involvement of co-regulators and repressors in miRNA-mediated SUMF1 expression in breast cancer cells and reveal the therapeutic potential of SUMF1 in endocrine-related malignancies

Differential desulfurization of dibenzothiophene by newly identified MTCC strains: Influence of Operon Array.

Since the sulfur specific cleavage is vital for the organic sulfur removal from fossil fuel, we explored potential bacterial strains of MTCC (Microbial Type Culture Collection) to desulfurize the Dibenzothiophene (DBT) through C-S bond cleavage (4-S pathway). MTCC strains Rhodococcus rhodochrous (3552), Arthrobacter sulfureus (3332), Gordonia rubropertincta (289), and Rhodococcus erythropolis (3951) capable of growing in 0.5 mM DBT were examined for their desulfurization ability. The presence of dsz genes as well as the metabolites was screened by polymerase chain reaction (PCR) and HPLC, respectively. All these strains showed > 99% DBT desulfurization with 10 days of incubation in minimal salt medium. From the HPLC analysis it was further revealed that these MTCC strains show differences in the end metabolites and desulfurize DBT differently following a variation in the regular 4-S pathway. These findings are also well corroborating with their respective organization of dszABC operons and their relative abundance. The above MTCC strains are capable of desulfurizing DBT efficiently and hence can be explored for biodesulfurization of petrochemicals and coal with an eco-friendly and energy economical process

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Possible bacterial strains containing <i>dszABC</i> operon.

<p>Possible bacterial strains containing <i>dszABC</i> operon.</p

List of organisms comparing their desulfurizing efficiency in percentage removal of sulfur from literature with reference to the present study.

<p>List of organisms comparing their desulfurizing efficiency in percentage removal of sulfur from literature with reference to the present study.</p

4S-Pathway of biodesulfurization of dibenzothiophene (DBT).

<p>Four enzymes (DszA, DzsB, DszC and DszD) are involved in the pathway where the first three steps catalyzed by flavin mononucleotide reduced (FMNH₂)-dependent monooxygenases, those leading to DBT-sulfoxide (DBTO), DBT-dioxide (DBTO₂) and hydroxyphenyl benzenesulfinate (HPBS), respectively. The final desulfurization step to 2-hydroxybiphenyl (2-HBP) is catalyzed by desulfinase.</p

Screening of <i>dsz</i> genes in different MTCC strains <i>Rhodococcus rhodochrous</i> (3552), <i>Artrobacter sulfureus</i> (3332), <i>Gordonia rubropertincta</i> (289) and <i>Rhodococcus erythropolis</i> (3951) enriched with DBT.

<p>The different lanes are showing the amplified products of different genes <i>dszA</i>, <i>dszB</i>, <i>dszC</i> and <i>dszD</i>.</p

Standardization of PCR product of <i>dsz</i> genes and 16s rRNA with different annealing temperatures.

<p>The PCR products with different annealing temperature were electrophoresed in agaraose gel against 100 bp DNA ladder.</p

Differential desulfurization of dibenzothiophene by newly identified MTCC strains: Influence of Operon Array - Fig 4

<p>HPLC graph showing the retention time of (a) DBT and (b) 2-HBP. (c) The control DBT without microorganism also shows same retention time with respect to the standard DBT without any degradation.</p